Neural stem cell-conditioned medium ameliorates Aβ25-35-induced damage in SH-SY5Y cells by protecting mitochondrial function

Abstract

Inhibition of amyloid β (Aβ)-induced mitochondrial damage is considered crucial for reducing the pathological damage in Alzheimer's disease (AD). We evaluated the effect of neural stem cell-conditioned medium (NSC-CDM) on Aβ25-35-induced damage in SH-SY5Y cells. An in vitro model of AD was established by treating SH-SY5Y cells with 40 µM Aβ25-35 for 24 h. SH-SY5Y cells were divided into control, Aβ25-35 (40 µM), Aβ25-35 (40 µM) + NSC-CDM, and Aβ25-35 (40 µM) + neural stem cell-complete medium (NSC-CPM) groups. Cell viability was detected by CCK-8 assay. Apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) were detected by flow cytometry. Malondialdehyde content was detected by ELISA assay. Western blot analysis was used to detect cytochrome c release and apoptosis-related proteins. Transmission electron microscopy was used to observe mitochondrial morphology. Cell viability significantly decreased and apoptosis significantly increased in SH-SY5Y cells treated with Aβ25-35, and both effects were rescued by NSC-CDM. In addition, NSC-CDM reduced ROS production and significantly inhibited the reduction of MMP caused by Aβ25-35. Furthermore, NSC-CDM ameliorated Aβ25-35-induced reduction in Bcl-2 expression levels and increased the expression levels of cytochrome c, caspase-9, caspase-3, and Bax. Moreover, Aβ25-35 induced the destruction of mitochondrial ultrastructure and this effect was reversed by NSC-CDM. Collectively, our findings demonstrated the protective effect of NCS-CDM against Aβ25-35-induced SH-SY5Y cell damage and clarified the mechanism of action of Aβ25-35 in terms of mitochondrial maintenance and mitochondria-associated apoptosis signaling pathways, thus providing a theoretical basis for the development of novel anti-AD treatments.

FIGURE 1

Different concentration and time of Aβ_25–35 treatment in SH-SY5Y cells. (A) CCK-8 detection of Aβ_25–35 (30 μM) treatment in SH-SY5Y cells for 12, 24, 36, and 48 h. (B) CCK-8 detection of Aβ_25–35 (0, 10, 20, 30, 40, and 50 mM) treatment in SH-SY5Y cells for 24 h. *p < 0.05, **p < 0.01. When the time was increased to 36 h, the cell viability decreased to 58.62 ± 1.26% compared with the control group. Different concentrations of Aβ_25–35 were then used to treat SH-SY5Y cells for 24 h. As the concentration of Aβ_25–35 increased, the cell survival rate decreased gradually. At 40 μM, the cell viability decreased to 56.62 ± 1.26% compared with the control group.

FIGURE 2

Neural stem cell-conditioned medium (NSC-CDM) protected against Aβ_25–35-induced toxicity, including decreased cell viability and increased apoptosis, in SH-SY5Y cells. (A) The cell viability of SH-SY5Y cells was evaluated by CCK-8 assay in the control, Aβ_25–35 (40 μM), Aβ_25–35 (40 μM) + neural stem cell-complete medium (NSC-CPM), and Aβ_25–35 (40 μM) + NSC-CDM groups for 24 h. (B and C) The apoptotic rates of the cells were labeled with the TUNEL assay in the control, Aβ_25–35 (40 μM), Aβ_25–35 (40 μM) + NSC-CPM, and Aβ_25–35 (40 μM) + NSC-CDM groups for 24 h. The white arrow represents apoptotic cells. Scale bar = 100 mm. (D) Apoptotic cells were examined by annexin V-FITC/PI double-staining in the control, Aβ_25–35 (40 μM), Aβ_25–35 (40 μM) + NSC-CPM, and Aβ_25–35 (40 μM) + NSC-CDM groups for 24 h. *p < 0.05, **p < 0.01, ***p < 0.001. Aβ_25–35 significantly decreased the cell viability of SH-SY5Y cells as compared with the control group. In contrast, both NSC-CPM and NSC-CDM had an inhibitory effect on the cytotoxicity induced by Aβ_25–35, with the NSC-CDM group demonstrating a higher cell viability than the NSC-CPM group. Next, we applied TUNEL and Annexin V-FITC/PI double-staining to determine the number of apoptotic SH-SY5Y cells. The nuclear fragmentation that is a characteristic feature of apoptotic cells was clearly observed in the Aβ_25–35-induced SH-SY5Y cells. In addition, condensed nuclei were also identified. However, the numbers of TUNEL-positive nuclei were significantly lower in the NSC-CPM or NSC-CDM + Aβ_25–35-treated cells, with the NSC-CDM group demonstrating a lower number of TUNEL-positive nuclei than the NSC- CPM group. In addition, cellular apoptosis was examined by Annexin V-FITC/PI double-staining and showed the same trends.

FIGURE 3

Neural stem cell-conditioned medium (NSC-CDM) protected against mitochondrial pathway-related apoptosis in SH-SY5Y cells induced by Aβ_25–35. (A) Flow cytometry was used to detect the relative intensity of reactive oxygen species (ROS) in the SH-SY5Y cells in the control, Aβ_25–35 (40 μM), Aβ_25–35 (40 μM) + NSC-CPM, and Aβ_25–35 (40 μM) + NSC-CDM groups for 24 h. (B) The MDA contents of the SH-SY5Y cells were assessed by ELISA kits in the control, Aβ_25–35 (40 μM), Aβ_25–35 (40 μM) + NSC-CPM, and Aβ_25–35 (40 μM) + NSC-CDM groups for 24 h. (C) Changes in the MMP by JC-1 in the control, Aβ_25–35 (40 μM), Aβ_25–35 (40 μM) + NSC-CPM, and Aβ_25–35 (40 μM) + NSC-CDM groups for 24 h. (D) Cytochrome c, caspase-9, caspase-3, Bcl-2-associated X protein (Bax), and B-cell lymphoma 2 (Bcl-2) protein expressions were evaluated by Western blot analysis in the control, Aβ_25–35 (40 μM), Aβ_25–35 (40 μM) + NSC-CPM, and Aβ_25–35 (40 μM) + NSC-CDM groups for 24 h. *p < 0.05, **p < 0.01, ***p < 0.001. The ROS content of the Aβ_25–35 group was significantly higher than that of the control group. The ROS content in the NSC-CPM and NSC-CDM+Aβ_25–35 group was significantly lower than that in the Aβ_25–35 group. The results of MDA content assays were similar to those of ROS assays. In addition, a decrease in MMP became obvious in the Aβ_25–35 group. These effects were reversed by the administration of NSC-CPM or NSC-CDM. The results from the Western blot analysis showed a decrease in the Bcl-2 expression level and an increase in the cytochrome c, caspase-9, caspase-3, and Bax expression levels in the Aβ_25–35 group compared with the control group.

FIGURE 4

Transmission electron microscopy was used to detect the ultrastructure of the mitochondria in the SH-SY5Y cells in the control, Aβ_25–35 (40 μM), Aβ_25–35 (40 μM) + neural stem cell-conditioned medium (NSC-CPM), and Aβ_25–35 (40 μM) + NSC-CDM groups for 24 h. The black arrow indicates normal mitochondria, whereas the white arrow indicates swollen mitochondria. Scale bar = 2 mm. Mitochondrial swelling was observed in the SH-SY5Y cells treated with Aβ_25–35. In particular, the crista of the mitochondria was observed to almost disappear or disintegrate. However, in the NSC-CPM and NSC-CDM+Aβ_25–35 groups, although most of the mitochondria were swollen and disrupted, we observed some normal mitochondria with the crista split. In addition, mitochondrial swelling in the NSC-CDM + Aβ_25–35 group was considered mild compared with that of the NSC-CPM + Aβ_25–35 group.